The interactions between transcription factors (TFs) and their DNA binding sites are an integral part of the gene regulatory networks within cells. Although many TFs have had their DNA binding specificities determined in recent years in various organisms, and while combinatorial regulation of gene expression by TFs is well known in animal development, we still have a poor understanding of how protein-protein interactions (PPIs) affect TF DNA binding specificities and effects on the expression of their target genes. In this project, we will focus on Drosophila melanogaster as a convenient, experimentally powerful model organism with significant transcriptional regulatory complexity. Specifically, we will investigate: (1) what TFs physically interact with each other, such as to form heterodimers? (2) do TFs have different DNA binding preferences individually versus as complexes? (3) do different TF interaction partners result in different binding properties of the TFs in vivo or associate with different biological processes? Altogether, these studies will lead to a better understanding of how PPIs among sequence-specific TFs affect TF regulatory specificity. These studies also will provide a valuable resource of datasets for the community.